Surface-enhanced Raman spectroscopy (SERS) has proven to be one of the most sensitive methods available for trace chemical analysis by detecting single molecules (see Kneipp, K., Wang, Y., Dasari, R. R., and Feld, M. S., “Approach to Single-Molecule Detection Using Surface-Enhanced Resonance Raman Scattering (SERRS): A Study Using Rhodamine 6G on Colloidal Silver”, Applied Spectroscopy, 49, 780-784 (1995) or Nie, S. and S. R. Emory, “Probing Single Molecules and Single Nanoparticles by Surface Enhanced Raman Scattering”, Science, 275, 1102 (1997)). In addition to sensitivity, the rich molecular vibrational information provided by Raman scattering yields exceptional specificity and allows identifying virtually any chemical as well as distinguishing multiple chemicals in mixtures (see Garrel, R. L., “Surface-Enhanced Raman Spectroscopy,” Analytical Chemistry, 61, 401A-411A (1989) or Storey, J. M. E., Barber, T. E., Shelton, R. D., Wachter, E. A., Carron, K. T., and Jiang, Y. “Applications of Surface-Enhanced Raman Scattering (SERS) to Chemical Detection”, Spectroscopy, 10(3), 20-25 (1995)). SERS involves the absorption of incident laser photons, generating surface plasmons within nanoscale metal structures, which then couple with nearby molecules (the analyte chemical), to thereby enhance the efficiency of Raman scattering by six orders of magnitude or more (Jeanmaire, D. L., and R. P. Van Duyne, “Surface Raman Spectroelectrochemistry”, J. Electroanal. Chem., 84, 1-20 (1977) or Weaver, M. J., Farquharson, S., Tadayyoni, M. A., “Surface-enhancement factors for Raman scattering at silver electrodes. Role of adsorbate-surface interactions and electrode structure”, J. Chemical Physics, 82, 4867-4874 (1985)).
Previous research has employed primarily the three most common methods of generating SERS, using: (1) activated electrodes in electrolytic cells (see for example Jeanmaire or Weaver, supra); (2) activated silver and gold colloid reagents (Kerker, M., O. Siiman, L. A. Bumm, D.-S. Wang, “Surface-enhanced Raman Scattering of citrate ion adsorbed on colloidal silver”, Applied Optics, 19, 3253-3255 (1980) or Angel, S. M., L. F. Katz, D. D. Archibold, L. T. Lin, D. E. Honigs, “Near Infrared Surface-enhanced Raman Spectroscopy. Part II: Copper and gold colloids”, Applied Spectroscopy, 43, 367 (1989)); or (3) activated silver and gold substrates (Seki., H., “Surface-enhanced Raman Scattering of pyridine on different silver surfaces”, J. Chemical Physics, 76, 4412-4418 (1982) or Li, Y.-S., T. Vo-Dinh, D. L. Stokes, Y. Wang, “Surface-Enhanced Raman Analysis of p-Nitroaniline on Vacuum Evaporation and Chemical Deposited Silver-Coated Alumina Substrates”, Applied Spectroscopy, 46, 1354 (1992)).
However, none of the foregoing techniques is capable of providing sufficiently reproducible measurements to enable the use of SERS for quantitative analysis. This is largely due to the inability to reproducibly manufacture a surface-enhanced Raman-active medium. More specifically, the first technique referred to above uses electrodes that are “roughened” by changing the applied potential between oxidation and reduction states; it is found that the desired metal surface features cannot be reproduced faithfully from one roughening procedure to the next. In the second technique referred to, colloids are prepared by reducing a metal salt solution to produce metal particles, which in turn form aggregates. Particle size and aggregate size are strongly influenced by initial chemical concentrations, temperature, pH, and rate of mixing, and again therefore the desired features are not reproducible. Finally, the third technique mentioned uses substrates that are prepared by depositing the desired metal onto a surface having the appropriate roughness characteristics. To permit the analysis, the sample is preferably dried on the surface to concentrate the analyte on the active metal, and once again replication is difficult to achieve. The relative merits of the three methods for preparing SER-active surfaces, described above, have been further reviewed by K. L. Norrod, L. M. Sudnik, D. Rousell, and K. L. Rowlen in “Quantitative comparison of five SERS substrates: Sensitivity and detection limit,” Applied Spectroscopy, 51, 994-1001 (1997).
As disclosed by Farquharson et al. in U.S. Pat. No. 6,623,977 (issued Sep. 23, 2003 and of common assignment herewith, and published as International Publication No. WO 01/33189 A2, dated 10 May 2001), the entire specification of which is hereby incorporated by reference thereto, sol-gels have been developed to trap particles of silver or gold (or of certain other metals) to provide an improved medium for reproducibly generating surface-enhanced Raman (SER) scattering. It is appreciated that the particle size and aggregation state of the metal dopant are stabilized, once the sol-gel has formed, and that the sample and/or solvent will not alter the plasmon-generating capabilities of the trapped metal particles. Albeit changes in pH may still result in variable Raman signal intensities, such as in the case of weak acids and bases where the relative concentrations of the ionized and non-ionized forms may be influenced, Farquharson et al. have demonstrated reasonably reproducible measurements, whereby some 36 repeat measurements of the same chemical, using multiple glass vials coated with silver-doped sol-gels, yielded a standard deviation of ˜15% (Farquharson, S., Gift, A., Maksymiuk, P., Inscore, F., Smith, W., Morrisey, K., and Christesen, S. D., “Chemical agent detection by surface-enhanced Raman spectroscopy”, SPIE, 5269, 16-22 (2003). Transmission electron micrographs have shown however that the distribution of metal particles within the sol-gel is inhomogeneous, which will cause variation in the SER-activity, as a function of the position of the excitation laser focal spot on the sol-gel, and is a likely source of the observed variations of SER spectral band intensities.
Quantitative measurements are of course fundamental to analytical chemistry, and most instruments and methods currently employed require some form of calibration to ensure the accuracy of measurements made. In the case of Raman spectroscopy, the intensities of spectral bands, or peaks, present in the scans produced are directly proportional to the concentrations of the analytes being measured. A measurement of the intensity of such bands, as either peak height or peak area, for a chemical of known concentration can therefore be used to calculate the concentration of the same chemical in an unknown sample, by measuring its corresponding spectral band intensities. Variations in laser power, detector response, ambient temperature, etc. can however influence the intensity of the spectral bands and thereby introduce significant error in the quantitative calculation.
A useful method of overcoming such variation errors involves the inclusion of a chemical of known concentration in the unknown sample, and using its Raman spectral band intensity as a reference to the band intensity of the chemical of unknown concentration (Pelletier, M. J., Ed. “Analytical Applications of Raman Spectroscopy,” Blackwell Science Ltd., London, 1999, p. 20). The choice of internal reference chemical employed depends somewhat upon the nature of the sample that is to be measured, but it is important that the spectral bands of the reference chemical that are to be used for quantifying the concentration should not overlap the spectral bands of the unknown sample to a degree that would interfere with the quantitative calculation. The prior art does not address the variability of amount of Raman scattering enhancement produced by surface-enhanced Raman-active media and, in any event, does not disclose reference chemicals, or provide suitable referencing techniques, that are specific to SERS or to measurement and analytical methods based thereupon.